SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin

Biosynthesis in the Wheat Pathogen Parastagonospora nodorum

Yit-Heng Chooi,a,b Mariano Jordi Muria-Gonzalez,a Oliver L. Mead,a Peter S. Solomona
Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australiaa; School of Chemistry and
Biochemistry, University of Western Australia, Perth, Western Australia, Australiab

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat patho-
gen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829
(SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH pro-
duction. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus
nidulans. The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of
AOH in vitro and its absence in planta. Subsequent characterization of the ⌬SnPKS19 mutants showed that SnPKS19 and AOH
are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast
light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH syn-
thase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between
AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH
production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

M ycotoxins pose serious health risks to humans and animals

(1, 2). Consumption of food and feed contaminated with
mycotoxins can lead to various mycotoxicoses. Mycotoxins also
cause significant economic losses in agriculture due to reduced
crop value resulting from mycotoxin contamination and losses in
animal productivity from mycotoxin-induced health problems
(2). Furthermore, there are increasing concerns about mycotoxins
in indoor environments (3), the contamination of water with my-
cotoxins in agricultural land affected by mycotoxin-producing
plant pathogens (4), and the effect of climate change on myco-
toxin contamination of pre- and postharvest food (5, 6).
Deciphering the molecular genetic basis of mycotoxin produc-
tion in fungi will improve our understanding of its genetic regu-
lation and facilitate the identification of potential mycotoxin-pro-
ducing fungi in the environment (7, 8). There has been
considerable interest in developing PCR-based diagnosis methods
for detection of mycotoxin-producing fungi in agricultural com-
modities and in food and feed (9). Such diagnosis will also help
determine if a fungal strain is suitable for use in the food industry
or as a biocontrol agent. With the wide availability of genomic
technologies, the genome of a fungal strain can be rapidly scanned
for potential genes encoding mycotoxin biosynthesis. The genetic
and molecular bases for biosynthesis of several important myco-
toxins are well known (10), such as those for aflatoxins (11, 12),
fumonisins (13, 14), zearalenone (15, 16), ergot alkaloids (17, 18),
and trichothecenes (19, 20). Nonetheless, the molecular genetic
bases for the production of many other mycotoxins, such as
ochratoxins, altertoxins, and alternariol, remain elusive.
Alternariol (AOH) and alternariol-9-methyl ether (AME) are
two important mycotoxins that are well known to be produced by
fungi in the Alternaria genus, which are capable of infecting a
variety of crop plants. The two mycotoxins are common contam-
inants of food such as grain and grain-based products, fruits/fruit
juice, and vegetable products (in particular, tomato products)
(21–23). They have been reported to exhibit cytotoxic, fetotoxic,
teratogenic, and possible mutagenic and estrogenic effects (24–27)
and may contribute to development of human esophageal cancer
(28). AOH has been demonstrated to inhibit cholinesterase (29)
and topoisomerases I and II (30). Furthermore, it was shown that
AOH induces DNA damage and cell cycle arrest in vitro in the
murine macrophage RAW 264.7 cell line, and the treated cells
exhibited an abnormal nuclear morphology (31, 32).
Recently, AOH was reported to be produced by Parastagono-
pora nodorum (synonymous to Stagonospora nodorum and Pha-
eosphaeria nodorum), an important wheat pathogen in Australia
and worldwide. AOH was first detected in P. nodorum in a
metabolomics study of a mutant lacking a short-chain dehydro-
genase gene, Sch1, which accumulated AOH at a 200-fold-greater
concentration than the wild type (WT) (33). Sch1 was demon-
strated to be regulated by G-protein signaling and is required for
asexual sporulation (34). Subsequently, a transcription factor
gene, StuA, has also been shown to be a positive regulator of AOH
production (35). The discovery of AOH in P. nodorum raised new
concerns about the health implication of this wheat pathogen.
Two polyketide synthase (PKS) genes have been recently in-
ferred in an RNA-silencing knockdown study to be involved in
AOH biosynthesis in Alternaria alternata (36). In this study, we
Received 27 January 2015 Accepted 20 May 2015
Accepted manuscript posted online 29 May 2015
Citation Chooi Y-H, Muria-Gonzalez MJ, Mead OL, Solomon PS. 2015. SnPKS19
encodes the polyketide synthase for alternariol mycotoxin biosynthesis in the
wheat pathogen Parastagonospora nodorum. Appl Environ Microbiol
81:5309 –5317. doi:10.1128/AEM.00278-15.
Editor: A. A. Brakhage
Address correspondence to Yit-Heng Chooi, yitheng.chooi@uwa.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.00278-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.00278-15

August 2015 Volume 81 Number 16 Applied and Environmental Microbiology aem.asm.org 5309
Chooi et al.
have unequivocally identified the PKS gene responsible for the
production of AOH in P. nodorum by both targeted genetic dis-
ruption and heterologous expression. The P. nodorum AOH PKS
gene is different from that identified previously but is highly con-
served with another PKS gene in A. alternata. Furthermore, iden-
tification of the P. nodorum PKS AOH gene has enabled the survey
of homologs in other fungal genomes.
MATERIALS AND METHODS
P. nodorum strains and culturing conditions. The wild-type P. nodorum
strain SN15 was obtained from Department of Agriculture and Food
Western Australia (DAFWA) and had been previously deposited in Amer-
ican Type Culture Collection (ATCC MYA-4574) and the Fungal Genet-
ics Stock Center (FGSC 10173). Both the wild-type and mutant strains
generated in this study were maintained on V8-supplemented potato dex-
trose agar (V8-PDA) plates at 22°C under a 12-h dark/light regime, which
induces sporulation (37). For screening of AOH and AME production in
P. nodorum wild-type strain SN15 and ⌬SnPKS19 mutants, the strains
were grown in defined minimal medium agar (MMA) (37) in the dark at
22°C for 7 days.
Aspergillus nidulans strain TNO2A3 (from the Fungal Genetics Stocks
Centre, FGSC A1149) was maintained on Aspergillus glucose minimal
medium (GMM) agar (38) supplemented with 0.5 mM pyridoxine, 0.01
␮g/ml riboflavin, 5 mM uridine, and 5 mM uracil. Aspergillus nidulans
TNO2A3 and the transformed mutants were cultured in liquid GMM at
28°C with shaking 150 rpm.
Metabolite profile analysis. For the detection of AOH and AME in the
P. nodorum SN15 extracts from various growth conditions, liquid chro-
matography-mass spectrometry (LC-MS) was performed on an Agilent
1200 LC system (Agilent, Santa Clara, CA, USA) coupled to an Agilent
6520 quadrupole time of flight (QToF) system with a Jetstream electros-
pray ionization (ESI) source to achieve higher sensitivity (see the methods
in the supplemental material).
For comparison of the metabolite profiles of P. nodorum ⌬SnPKS19
strains against that of the wild type, agar blocks cut out from a 10-day-old
MMA culture were extracted with an approximately equal volume of ethyl
acetate (EtOAc). For A. nidulans expressing SnPKS19, 10 ml of liquid
GMM cultures was sampled at 48 and 72 h postinoculation and extracted
with an equal volume of EtOAc. The extracts were dried in vacuo and
redissolved in methanol (MeOH). The metabolite profile analyses were
performed on an Agilent 1200 LC system coupled to a diode array detector
(DAD) and an Agilent 6120 quadrupole MS with an ESI source. Chro-
matographic separation and conditions were exactly as described previ-
ously (39). The authenticity of AOH was confirmed by comparing the m/z
ratio, UV spectrum, and retention time with those of the corresponding
commercial standards (LGC standards).
Transformation and screening of P. nodorum ⌬SnPKS19 mutants.
A double-homologous SnPKS19 knockout (KO) cassette carrying a hy-
gromycin resistance marker was assembled using the Gibson assembly
method (40) on a pGEM-T easy vector backbone (Promega). The hygro-
mycin resistance marker was amplified from pAN7-1 (41) using GPE1-F
and TtrpC-R primers (see Table S1 in the supplemental material). The 5=
and 3= homologous regions of SnPKS19 (1.6 kbp each) were amplified
with the SnPKS19-KO-P2/P3 and SnPKS19-KO-P4/P5 primer pairs, re-
spectively. The three overlapping DNA fragments and the linearized vec-
tor backbone were assembled using the Gibson assembly master mix
(NEB) following the manufacturer’s protocol. The resulting plasmid,
pGEM-SnPKS19-KO, was sequenced to confirm the construct. Finally,
the 5.8-kbp SnPKS19 KO cassette (see Fig. S3 in the supplemental mate-
rial) was liberated by digesting the plasmid with NotI enzyme (NEB) and
gel purified for P. nodorum transformation. Transformation of P. nodo-
rum with the SnPKS19 KO cassette was achieved by the polyethylene gly-
col (PEG)-mediated protoplast transformation protocol as described pre-
viously (42). The integration of the KO cassette in the correct locus was
confirmed by diagnostic PCR using two pairs of primers, SnPKS19-KO-
5310 aem.asm.org Applied and Environmental Microbiology
P1/Hyg-N-R and Hyg-C-F/SnPKS19-KO-P6, as described previously
(43). The deletion of SnPKS19 was further confirmed by Southern blot-
ting using a digoxigenin (DIG) DNA labeling and detection kit (Roche)
according to the manufacturer’s instructions.
Characterization of the virulence and oxidative stress tolerance of P.
nodorum ⌬SnPKS19 mutants. A modified detached-leaf assay (DLA)
was used to assess the virulence of P. nodorum ⌬SnPKS19 mutants against
wheat. The protocol was as described previously (44), with the exception
of using 0.25% gelatin instead of 0.2% Tween 20 for spore suspension. The
oxidative tolerance of P. nodorum ⌬SnPKS19 mutants compared to wild
type was determined by placing a 5-mm-diameter agar block on MMA
containing 0, 2, 5, and 10 mM hydrogen peroxide in triplicates, as de-
scribed previously (45).
Cloning and heterologous expression of SnPKS19 in Aspergillus ni-
dulans. The original annotations of the SnPKS19 translated protein in the
GenBank and JGI databases lack the N-terminal starter unit:acyl carrier
protein (ACP) transacylase (SAT) domain. The missing SAT domain con-
served among nonreducing PKSs (NR-PKSs) was located by TBLASTX
analysis with an additional 1,000 bp upstream of the SnPKS19 coding
region added as a query sequence. The SAT domain was restored by man-
ual annotation with the corrected annotation, placing the new ATG start
codon 656 bp upstream of the original start codon.
SnPKS19 was cloned into the pBARGPE1-LIC expression plasmid.
pBARGPE1-LIC was modified from pBARGPE1 (from FGSC) for liga-
tion-independent cloning (LIC) as described previously (46). The plas-
mid contains an gpdA promoter and trpC terminator for gene expression
and a bar gene for glufosinate resistance. The revised full-length SnPKS19
coding region, including introns (see Fig. S5 in the supplemental mate-
rial), was PCR amplified from P. nodorum SN15 genomic DNA using
primers LIC-SnPKS19-F_new and LIC-SnPKS19-R (see Table S1 in the
supplemental material). DNA assembly using LIC was performed as de-
scribed previously (46). Transformation of A. nidulans TNO2A3 with the
resulting plasmid, pBGP-SnPKS19, was performed using the PEG-medi-
ated transformation protocol as described previously (47). For selection
of glufosinate-resistant transformants, the glufosinate was prepared from
the commercial herbicide Basta (Bayer CropScience) as described previ-
ously and added at 25 ␮l/ml of Aspergillus glucose minimal medium with
10 mM ammonium tartrate as the sole nitrogen source (48).
The transcription of SnPKS19, pkgA (ANIA_07071), and pkgB
(ANIA_07070) in the A. nidulans strain transformed with pBG-SnPKS19
was checked using reverse transcriptase PCR (RT-PCR), as described pre-
viously (46), with primer pairs SnPKS19-RT-F/R, AN7071-RT-F/R,
AN7070-RT-F/R, which flank the last (3=-end) introns in the three respec-
tive genes (see Table S1 in the supplemental material).
Phylogenetic analysis. The conserved ␤-keto-acyl synthase (KS) do-
main sequences were used to infer the phylogenetic relationship between
SnPKS19 and other fungal NR-PKSs. A total of 19 NR-PKSs characterized
in previous studies were included as reference sequences, while seven
additional characterized highly reducing PKSs (HR-PKSs)/PKS-NRPSs
were included as outgroup (see Table S2 in the supplemental material). To
investigate the distribution of AOH and norlichexanthone (NLX) syn-
thases, the phylogenetic analysis further included all NR-PKSs with KS
domains that shared ⬎80% identity with SnPKS19 from the NCBI
GenBank and JGI Ascomycota (http://genome.jgi-psf.org/ascomycota
/ascomycota.info.html) databases. Multiple-protein alignment was per-
formed using CLUSTALX, and the resulting alignment was trimmed (cor-
responds to amino acids 418 to 821 of the corrected SnPKS19 protein
sequence). The phylogenetic analysis was performed using the neighbor-
joining method (49) embedded in the Geneious software 7.17 software
(Biomatters Ltd.). The tree was constructed with 1,000 bootstrap repli-
cates, and branches corresponding to partitions that were reproduced in
fewer than 50% of bootstrap replicates are collapsed.
Nucleotide sequence accession number. The corrected annotation
for SnPKS19 has been deposited in GenBank under accession no.
KP941080.
August 2015 Volume 81 Number 16

FIG 1 Comparison of metabolite profiles of the P. nodorum wild-type (WT)
strain and ⌬SnPKS19 mutants grown on minimal medium agar. UV chro-
matograms of P. nodorum WT/ectopic mutant and ⌬SnPKS19 mutants are
shown in comparison to AOH and AME standards.
RESULTS
Detection of alternariol under various growth conditions. We
first investigated for growth conditions that favor AOH produc-
tion in the P. nodorum SN15 wild-type strain. The strain was cul-
tured on different media and under different growth conditions
and checked for AOH production by LC-DAD-MS. P. nodorum
produced AOH (m/z 259) when grown in defined minimal agar
medium (MMA) at 22°C after 7 days (Fig. 1). AOH could also be
FIG 2 Comparison of the SnPKS19 domain architecture with those of other nonreducing polyketide synthases (NR-PKSs). SAT, starter unit:ACP transacylase
domain; KS, ␤-keto-acyl synthase domain; MAT, malonyl-CoA:ACP transferase domain; PT, product template domain; ACP, acyl carrier protein domain;
TE/CLC, thioesterase/Claisen cyclase domain; M␤L-TE, metallo-␤-lactamase-like thioesterase. M␤L-TE is a discrete protein.
August 2015 Volume 81 Number 16 Applied and Environmental Microbiology
Alternariol Synthase from Parastagonospora nodorum
detected in shaking P. nodorum Fries medium cultures (see the
methods and Fig. S1 in the supplemental material). To detect the
possible production of AOH in planta, two experiments were per-
formed, i.e., detached-leaf assay (DLA) and in vivo glume infec-
tion assay (see the methods in the supplemental material). How-
ever, we failed to detect any AOH in either the infected wheat
leaves from DLAs or the infected wheat grains from glume infec-
tion assays (see Fig. S1 in the supplemental material).
Bioinformatic analysis of putative PKS genes in P. nodorum.
We previously performed a detailed survey of the P. nodorum
genome for secondary metabolite genes and have compiled the
polyketide synthase (PKS) gene inventory for this wheat pathogen
(50). To identify the PKS gene encoding AOH in P. nodorum, we
first searched for P. nodorum PKSs that are homologous to PksH
and PksJ, which were previously implied to be involved in AOH
production in A. alternata by RNA interference (RNAi) silencing
experiments (36). However, a BLASTP search did not identify any
close homolog of A. alternata PksH and PksJ in P. nodorum.
To further expand the search for the AOH PKS, a genome-wide
comparison of all the PKSs in P. nodorum and A. alternata (36)
identified another PKS from A. alternata (PksI) sharing significant
homology (86% identity) to SNOG_15829 (abbreviated as
SnPKS19) in P. nodorum. SnPKS19 is a typical nonreducing PKS
(NR-PKS) (51, 52), consisting of a SAT domain, a ␤-keto-acyl
synthase (KS) domain, a malonyl coenzyme A (malonyl-CoA):
ACP transferase (MAT) domain, a product template (PT) do-
main, and an acyl carrier protein (ACP) domain. The original
annotation of SnPKS19 in the GenBank and JGI databases lacked
the SAT domain, but this N-terminal domain was restored by
manual annotation (Fig. 2; see Fig. S4 in the supplemental mate-
rial). PksI from A. alternata, however, is truncated at the 3= end
based on the annotation by Saha et al., and the predicted PKS
protein lacks the C-terminal ACP domain (Fig. 2) (36). The clos-
est characterized homolog for the two PKSs in the GenBank pro-
tein database is the norlichexanthone (NLX) synthase (GsfA) in
the griseofulvin pathway in Penicillium aethiopicum (synonym,
Penicillium lanosocoeruleum) (53, 54), which share high percent-
ages of protein identity (overall protein, 65% identity; KS domain,
83% identity). Both AOH and NLX are derived from a heptaketide
aem.asm.org 5311
Chooi et al.
FIG 3 Biosynthesis of alternariol (AOH) in comparison to norlichexanthone
(NLX) by polyketide synthases.
chain and involve a C-8 –C-13 aldol cyclization in the biosynthe-
sis, but the regiospecificity of the second cyclization diverges (Fig.
3). As AOH and NLX are structural isomers, we reasoned that it is
highly likely that the PKS gene SnPKS19 may encode the biosyn-
thesis of AOH.
The genes in the vicinity of SnPKS19 encode two putative
O-methyltransferases, a putative ␤-lactamase, a putative short-
chain dehydrogenase, a putative regulatory protein, and a pu-
tative transporter (see Fig. S2 in the supplemental material).
We extracted the expression profiles of SnPKS19 and its neigh-
boring genes from previous microarray data (55, 56). The pro-
duction of AOH in P. nodorum MMA culture and its absence in
DLA samples correlated with the expression of SnPKS19 during
the growth in vitro and its silence in planta (see Fig. S2 in the
supplemental material). One of the putative O-methyltrans-
ferase genes (SNOG_15830), along with the colocated regula-
tory protein (SNOG_15831) and the putative short-chain de-
hydrogenase (SNOG_15832) genes, appears to be coregulated
with SnPKS19, all with higher expression in vitro at 16 days
postinoculation (dpi) than at 4 dpi, and all were silent in
planta. Despite the presence of the putative O-methyltrans-
ferase gene in the gene cluster, we did not detect any O-meth-
ylated derivative of AOH in P. nodorum MMA culture (Fig. 1)
and under the various other growth conditions tested.
SnPKS19 is required for alternariol production in P. nodo-
rum. To confirm that the SnPKS19 gene is involved in AOH
biosynthesis, P. nodorum ⌬SnPKS19 mutants were generated by
polyethylene glycol (PEG)-mediated transformation with an
SnPKS19 knockout cassette. Diagnostic PCR identified three pos-
itive transformants where SnPKS19 had been disrupted by dou-
ble-homologous crossover recombination and replaced with a hy-
gromycin resistance marker (see Fig. S3 in the supplemental
material). The deletion of SnPKS19 in two transformants
(SnPKS19-KO2 and -KO3) was further confirmed by Southern
blotting (see Fig. S4 in the supplemental material). No observable
5312 aem.asm.org Applied and Environmental Microbiology
growth defect or differences in the growth rate between the mu-
tants and the wild-type (WT) P. nodorum were observed. The
colony morphology of the mutants on V8-PDA and minimal me-
dium plates were also indistinguishable from that of the WT.
Five transformants, including three ⌬SnPKS19 mutants and
two ectopic mutants, were selected and grown on MMA under the
above-mentioned condition. Comparative LC-MS metabolite
profile analysis of the EtOAc extracts from the cultures showed
that production of AOH was abolished in all the ⌬SnPKS19 mu-
tants but remained present in both ectopic mutants and the WT
(Fig. 1). Thus, this confirmed that SnPKS19 is required for AOH
biosynthesis in P. nodorum.
SnPKS19 alone is sufficient for biosynthesis of alternariol.
The predicted PKS protein encoded by SnPKS19 lacks the
polyketide product-releasing C-terminal thioesterase (TE)/
Claisen cyclase (CLC) domain commonly found in fungal NR-
PKSs (57, 58). Previous biosynthetic studies have shown that some
fungal NR-PKSs lacking a releasing domain require an additional
discrete metallo-␤-lactamase-like TE (M␤L-TE) protein to facil-
itate polyketide chain release or cyclization (Fig. 2) (54, 59–61).
Thus, the putative ␤-lactamase gene (SNOG_15826) in the vicin-
ity of SnPKS19 may be required for releasing of AOH via lactone
formation. However, upon close inspection, SNOG_15826 is seen
to share little similarity to the characterized M␤L-TEs, as it be-
longs to a different family of ␤-lactamases. Furthermore, the pre-
vious microarray data showed that SNOG_15826 was not coregu-
lated with the PKS gene SnPKS19 (see Fig. S3 in the supplemental
material), which supported its noninvolvement in AOH biosyn-
thesis.
To determine if SnPKS19 alone was sufficient for AOH synthe-
sis, SnPKS19 with the restored 5= region was cloned into plasmid
pBARGPE1-LIC for heterologous expression. The resulting plas-
mid, pBG-SnPKS19, was used to transform A. nidulans strain
TNO2A3 (from FGSC). Two glufosinate-resistant transformants
were verified to contain the SnPKS19 gene by PCR and were cul-
tured in Aspergillus minimal liquid medium for 72 h at 28°C. LC-
DAD-MS analysis of the EtOAc extracts from the cultures showed
that both transformants produced a unique peak with m/z (M ⫹
H)⫹ of 259, corresponding to AOH, which was absent in the A.
nidulans control strain transformed with the empty pBARGPE1-
LIC plasmid (Fig. 4). The result showed that SnPKS19 alone is
sufficient for biosynthesis of AOH in A. nidulans.
In a previous study, promoter replacement of the NR-PKS
gene pkgA (ANIA_07071) and the M␤L-TE gene pkgB (ANIA_
07070) in A. nidulans resulted in production of AOH along with
three other isocoumarins. To rule out that these two genes are
involved in AOH production in the A. nidulans strains harboring
SnPKS19, we performed an RT-PCR to determine the expression
of SnPKS19 and the two genes at 72 h postinoculation. RT-PCR
showed that SnPKS19, but not pkgA and pkgB, was transcribed and
spliced in the A. nidulans/pBG-SnPKS19 strains (see Fig. S6 in the
supplemental material).
SnPKS19 is not involved in the virulence and oxidative stress
protection of P. nodorum. The ability of the ⌬SnPKS19 mutant
strains (SnPKS19-KO2 and -KO3) to cause disease was assessed
using detached-leaf assays. No significant difference in disease
symptoms was observed on detached wheat leaves sprayed with
spores of ⌬SnPKS19 mutants compared to the controls (wild-type
and ectopic integration strains) (see Fig. S7A in the supplemental
material).
August 2015 Volume 81 Number 16

FIG 4 Heterologous production of AOH in Aspergillus nidulans. A UV chro-
matogram (A) and an extracted ion chromatogram (EIC) (m/z 259) (B) of the
culture extracts from A. nidulans transformed with pBG-SnPKS19 in compar-
ison to a control and an AOH standard are shown.
Some secondary metabolites are known to possess antioxidant
activities and are thought to serve as protectants against oxidative
stress (62). However, we did not observe a significant change in
the ability to tolerate increasing concentration of hydrogen per-
oxide between ⌬SnPKS19 mutant strains and wild-type strain
SN15 (data not shown).
Alternatiol does not exhibit phytotoxic or antimicrobial ac-
tivities. The phytotoxic activity of AOH on wheat leaves was ex-
amined by leaf infiltration on whole plant wheat seedlings (see the
methods in the supplemental material). No necrosis was observed
on wheat leaves at concentrations up to 200 ␮g/ml of AOH (see
Fig. S7B in the supplemental material). The capacity of AOH to
affect seed germination was also assessed. Unlike what we had
observed for (R)-mellein in a previous study (39), AOH did not
inhibit the germination of wheat and barrel medic seeds (data not
shown). We have also tested the antimicrobial activity of AOH
against Escherichia coli, Saccharomyces cerevisiae, Zymoseptoria
tritici, and three environmental bacterial strains isolated from
field wheat samples (Bacillus cereus, a Flavobacterium sp., and Sph-
ingobacterium multivorum) (see the methods in the supplemental
material). AOH did not inhibit the growth of any of the bacterial
and fungal strains tested at concentrations up to 200 ␮g/ml.
Phylogenetic analysis can differentiate alternariol synthases
from other fungal PKSs. Previous studies have shown that fungal
NR-PKSs (without a C-methyltransferase domain) can be classi-
August 2015 Volume 81 Number 16 Applied and Environmental Microbiology
Alternariol Synthase from Parastagonospora nodorum
fied by phylogeny into five major groups plus two additional
groups that harbor a C-methyltransferase domain (59, 63). The
two studies showed that the phylogenetic grouping correlated
with the polyketide product chain length and cyclization regiose-
lectivity of NR-PKSs. To determine the phylogenetic relationship
of SnPKS19 with NLX synthase GsfA and in relation to other
NR-PKSs, three PKSs from each group of group I to IV NR-PKSs,
along with six PKS sequences from group V, were selected as ref-
erence sequences for phylogenetic analysis (see Table S2 in the
supplemental material). The phylogenetic tree showed that the
NR-PKSs were separated into the five respective groups (groups I
to V), as expected (Fig. 5).
GsfA and SnPKS19 along with their close homologs grouped
together with other group V NR-PKSs but formed a separate sub-
clade. Within this subclade, SnPKS19 and GsfA homologs were
further separated into two smaller subclades, referred to here as
the AOH and NLX subclades, respectively. Within the NLX sub-
clades, there are two other NR-PKSs that originated from known
NLX/griseofulvin-producing fungi, i.e., Penicillium canescens
(Penca1|243077) and Penicillium raistrickii (Penra1|289401) (Fig.
4) (64). Likewise, the AOH subclade is supported by NR-PKSs
from AOH producers, including A. alternata (PksI) and Talaro-
myces aculeatus (Penac1|509622) (65). These analyses suggest that
the NR-PKS homologs that fall in the AOH and NLX subclades
may produce the respective compounds.
DISCUSSION
Using both reverse genetics and heterologous expression, we re-
cently identified the P. nodorum PKS gene responsible for produc-
tion of R-(⫺)-mellein, which exhibited antigerminative activity
(39). Here, using a similar approach, we unequivocally demon-
strated that SnPKS19, which is highly similar to the A. alternata
pksI product (86% protein identity), is solely responsible for the
biosynthesis of AOH in P. nodorum. Saha et al. previously re-
ported the involvement of pksH and pksJ in AOH biosynthesis in
A. alternata based on RNAi silencing experiments (36). In contrast
to PksH and PksJ, which are HR-PKSs commonly involved in
biosynthesis of aliphatic compounds, both the SnPKS19 and PksI
genes encode an NR-PKS that is typically involved in biosynthesis
of aromatic polyketides (51). Curiously, the pksI in A. alternata is
annotated as a truncated PKS gene (Fig. 2) (36). Quantitative real-
time reverse transcriptase PCR (qRT-PCR) from this previous
study by Saha et al. showed that pksI was transcribed and that its
expression correlated to the timing of AOH production, suggest-
ing that pksI is unlikely to be a pseudogene. Nevertheless, pksI was
not investigated further (36). It is not uncommon that RNA si-
lencing could result in off-target effects (66–68). Given that the
highly conserved KS domain was targeted in the study by Saha et
al., it is possible that the transcription of other PKS genes was
knocked down as well due to the potential complementary nature
of the nucleotide sequences. For example, transcription of both
pksI and -J was silenced in the pksH knockdown strain (36), which
may also explain the observed reduced AOH production if pksI is
responsible for AOH production in A. alternata. It is possible that
the presumed truncation of the pksI coding sequence is due to
automated annotation error (like in the original SnPKS19 anno-
tation) or frameshift errors in sequencing. Based on our results
from this study, it is very likely that pksI is the gene responsible for
AOH production in A. alternata. Further investigation is required
to verify the identity of AOH synthase in A. alternata.
aem.asm.org 5313
Chooi et al.
FIG 5 Phylogeny and distribution of AOH synthase homologs in fungi in relation to other fungal NR-PKSs. KS sequences used to construct the phylogenetic tree
are listed in Table S2 in the supplemental material.
An NR-PKS gene (pkgA) from A. nidulans has been recently
reported to produce AOH along with a mixture of several hexa-
and heptaketide isocoumarin products, but only in the presence of
the partner M␤L-TE gene (pkgB) (59). The abundances of the
heptaketides citreoisocoumarin and dehydrocitreoisocoumarin
are comparable to that of AOH in the pkgA and -B overexpression
strain. Overexpression of the PKS gene pkgA alone by promoter
exchange did not result in any observable product (59). Here, we
used the A. nidulans system to characterize SnPKS19 by heterolo-
gous expression and showed that the SnPKS19-expressing strains
accumulated significant quantities of AOH at 72 h. No related
isocoumarins identified in the previous study by Ahuja et al. (59)
were detected in the SnPKS19-expressing A. nidulans strains, and
the involvement of pkgA and -B is ruled out based on the RT-PCR
results (see Fig. S6 in the supplemental material) and the absence
of AOH in the control strain. Thus, we reasoned that AOH is the
major and final product of SnPKS19, while AOH along with the
hexaketide isocoumarins may be aberrant or derailed products of
PkgA and -B, possibly due to overexpression of both genes at the
same level (a nonoptimal gene/enzyme ratio) (69).
The PT domain of NR-PKSs mediates the cyclization of nonre-
duced polyketide chains into various aromatic systems (52, 70).
All the isocoumarin products produced by PkgA and -B shared the
5314 aem.asm.org Applied and Environmental Microbiology
C-2–C-7 cyclized ring in AOH. In contrast, NLX shared the C-8 –
C-13 cyclized ring in AOH (Fig. 3). It is possible that the
polyketide biosynthesis in PkgA involved PT domain-controlled
C-2–C-7 cyclization followed by-product release via hydrolysis or
lactonization catalyzed by PkgB. AOH was likely formed in the
pkgA- and B-overexpressing strain via spontaneous aldol cycliza-
tion after polyketide release, whereas the PT domain of NLX and
AOH synthases is likely to mediate the C-8 –C-13 cyclization in-
stead; the two pathways diverged during the second ring cycliza-
tion (C-6 –C-1 Claisen cyclization or C-2–C-7 aldol cyclization)
to yield NLX or AOH, respectively.
Previous phylogenetics analysis showed that the NLX synthase
GsfA fell within the group V NR-PKSs (59). The present study
showed that GsfA grouped together with group V NR-PKSs as well
but formed a separate subclade with SnPKS19 and other putative
NLX/AOH synthases from the group V NR-PKSs that require a
discrete M␤L-TE for product release, which include ACAS (60),
AptA (71), VrtA (54), AdaA (61), PkgA (59), and EncA (72). Thus,
the branching between these two subgroups of group V NR-PKSs
could represent the point where the partnership with M␤L-TE
was lost in the NLX/AOH synthases. It appears that the NLX/AOH
synthases have evolved the ability to off-load PKS products by
Claisen cyclization or hydrolysis/lactonization without C-termi-
August 2015 Volume 81 Number 16

nal appended TEs or discrete M␤L-TEs. The molecular mecha-
nism and the functional domain responsible for the polyketide
release in these TE-less NLX/AOH synthases remain to be eluci-
dated.
Based on the phylogenetic analysis, SnPKS19 and GsfA can be
further divided into two smaller subclades consisting of putative
AOH and NLX synthases, respectively. Both subclades are sup-
ported by two additional NR-PKSs from known AOH or NLX
producers. Besides the high similarity shared between SnPKS19
and Penac1|509622 from the AOH/AME producer T. aculeatus,
the two PKS gene clusters also encode a homologous O-methyl-
transferase (SNOG_15830/Penac1|509623; 71% protein identity),
suggesting that these two enzymes are likely involved in methyl-
ation of AOH. Although we did not detect any AME in P. nodorum
under the growth conditions tested in this study, the ability of P.
nodorum to produce AME cannot be excluded. Based on the gene
cluster map reported by Saha et al., an O-methyltransferase is
encoded next to A. alternata pksI as well (36), but the correspond-
ing gene was not deposited in a public database and therefore
cannot be compared. Similarly, the gene clusters containing the
putative NLX synthase gene in the NLX/griseofulvin producers P.
canescens and P. raistrickii encode all the homologous enzymes in
the P. aethiopicum gsf gene cluster required for griseofulvin bio-
synthesis (53, 54). These data together strongly support that phy-
logenetic analysis can be used to predict whether an NR-PKS gene
is responsible for production of AOH or NLX using SnPKS19 and
GsfA as reference sequences.
It is difficult to determine the exact number of fungal genome
sequences deposited in the NCBI GenBank database (312 are as-
comycetes listed in NCBI Genome), but the search for AOH syn-
thase homologs in the JGI database (http://genome.jgi-psf.org
/ascomycota/ascomycota.info.html) included 259 ascomycetes.
Although the AOH synthase gene does not appear to be ubiqui-
tous, the orthologs are not restricted to a single ascomycete class
but distributed across Dothideomycetes, Leotiomycetes, Eurotio-
mycetes, and Sordariomycetes (Fig. 5; see Table S2 in the supple-
mental material). It is less certain whether the two PKSs (Botrytis
cinerea BcPKS14 and Zasmidium cellare Zasce1|61908) basal to the
NLX and AOH clades are responsible for production of NLX or
AOH. Interestingly, a dibenzopyrone botrallin, structurally re-
lated to AOH, has been reported from Botrytis allii (73, 74). There
is also at least one instance where a lichenized fungus was reported
to produce both AOH and NLX (75). Thus, the possible existence
of such NR-PKSs that have the promiscuity to produce both struc-
tural isomers cannot be discounted. AOH had also been reported
in an endophytic Colletotrichum sp. (76), but no close homolog of
AOH synthase has been found among the sequenced Colletotri-
chum genomes.
Due to their occurrence in plant-pathogenic Alternaria species,
AOH and AME had also been investigated for potential phyto-
toxic activities, but most reports are negative (77, 78). Interest-
ingly, AME has been reported to suppress cyanobacterial growth
by inhibiting the photosynthetic electron transport chain (79). In
this study, we showed that the virulence of ⌬SnPKS19 against
wheat leaves in vitro is comparable to that of the wild type; thus,
AOH is unlikely to play a role in pathogenicity. This is not surpris-
ing given that the transcription of SnPKS19 is silent in planta based
on previous microarray data and that we did not detect any AOH
in infected wheat leaves or grains. Likewise, we did not detect any
phytotoxic activity against wheat for AOH using leaf infiltration
August 2015 Volume 81 Number 16 Applied and Environmental Microbiology
Alternariol Synthase from Parastagonospora nodorum
and seed germination. We have tested AOH against common lab-
oratory microorganisms and environmental isolates of bacteria
and fungi as well, but we did not detect any growth-inhibitory
activity. Thus, the biological and ecological roles of AOH remain a
mystery.
AOHs are often detected in wheat, sorghum, and barley grains
contaminated with Alternaria fungi and pose a health risk to hu-
mans and animals (23). However, an analysis of P. nodorum-in-
fected grains in this study failed to detect any AOH. Furthermore,
there has been no prior report of AOH in P. nodorum-contami-
nated grains, suggesting that AOH mycotoxin production is not
part of the disease cycle. Given that P. nodorum has the genetic
potential to produce AOH and has been shown to produce AOH
under laboratory culture conditions, it cannot be discounted that
the fungus may produce AOH on wheat grain when the condi-
tions become favorable, especially in the face of changing climatic
environments. Recently, it has shown that AOH production in A.
alternata is affected by light conditions and partially regulated by
the blue-light receptor LreA (80). The identification of the PKS
gene encoding AOH production in this study opens up the oppor-
tunity for investigating the molecular genetic regulation of AOH
in P. nodorum and other AOH-producing fungi. This discovery
will also facilitate the assessment of the genetic potential for pro-
duction of AOH in other fungi and the development of molecular
diagnostic methods for detection and quantification of AOH-pro-
ducing fungi in agricultural commodities and in food and feed.
ACKNOWLEDGMENTS
Y.-H.C. is supported by an Australian Research Council Discovery Early
Career Researcher Award (DECRA) fellowship. M.J.M.-G. is a recipient of
an Australian Government Endeavor Award and a Mexican CONACyT
scholarship. P.S.S. is an Australian Research Council Future Fellow.
Y.-H.C. thanks Jack Elix for helpful discussion.
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